Title : Cloning, mutagenesis, and structural analysis of human
pancreatic alpha-amylase expressed in Pichia pastoris
Abstract :
- Human pancreatic alpha-amylase ( HPA ) was expressed in the methylotrophic yeast Pichia pastoris and two mutants (D197A and D197N) of a completely conserved active site carboxylic acid were generated
- All recombinant proteins were shown by electrospray ionization mass spectrometry ( ESI-MS) to be glycosylated and the site of attachment was shown to be Asn461 by peptide mapping in conjunction with ESI-MS
- Treatment of these proteins with endoglycosidase F demonstrated that they contained a single N-linked oligosaccharide and yielded a protein product with a single N-acetyl glucosamine (GlcNAc) , which could be crystallized
- Solution of the crystal structure to a resolution of 2.0 A confirmed the location of the glycosyl group as Asn461 and showed that the recombinant protein had essentially the same conformation as the native enzyme
- The kinetic parameters of the glycosylated and deglycosylated wild-type proteins were the same while the k(cat)/Km values for D197A and D197N were 10(6)-10(7) times lower than the wild-type enzyme
- The decreased k(cat)/Km values for the mutants confirm that D197 plays a crucial role in the hydrolytic activity of HPA , presumably as the catalytic nucleophile